This invention relates to materials and methods for the detection of mutations in targeted nucleic acids. More specifically, the invention provides nucleic acid molecules encoding a mismatch specific nuclease and methods of use of the enzyme that facilitate the genetic screening of hereditary diseases and cancer. The method is also useful for the detection of genetic polymorphisms.
Several publications are referenced in this application by numerals in parenthesis in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference in the present specification.
The sequence of nucleotides within a gene can be mutationally altered or xe2x80x9cmismatchedxe2x80x9d in any of several ways, the most frequent of which being base-pair substitutions, frame-shift mutations and deletions or insertions. These mutations can be induced by environmental factors, such as radiation and mutagenic chemicals; errors are also occasionally committed by DNA polymerases during replication. Many human disease states arise because fidelity of DNA replication is not maintained. Cystic fibrosis, sickle cell anemia and some cancers are caused by single base changes in the DNA resulting in the synthesis of aberrant or non-functional proteins.
The high growth rate of plants and the abundance of DNA intercalators in plants suggests an enhanced propensity for mismatch and frameshift lesions. Plants and fungi are known to possess an abundance of single-stranded specific nucleases that attack both DNA and RNA (9-14). Some of these, like the Nuclease xcex1 of Ustilago maydis, are suggested to take part in gene conversion during DNA recombination (15,16). Of these nucleases, S1 nuclease from Aspergillus oryzue (17), and P1 nuclease from Penicillium citrinum (18), and Mung Bean Nuclease from the sprouts of Vigna radiata (19-22) are the best characterized. S1, P1 and the Mung Bean Nuclease are Zn proteins active mainly near pH 5.0 while Nuclease xcex1 is active at pH 8.0. The single strandedness property of DNA lesions appears to have been used by a plant enzyme, SP nuclease, for bulky adduct repair. The nuclease SP, purified from spinach, is a singlestranded DNase, an RNase, and able to incise DNA at TC6-4 dimers and cisplatin lesions, all at neutral pH (23,24).
In Escherichia coli, lesions of base-substitution and unpaired DNA loops are repaired by a methylation-directed long patch repair system. The proteins in this multienzyme system include MutH, MutL and MutS (1, 2). This system is efficient; but the C/C lesion and DNA loops larger than 4 nucleotides are not repaired. The MutS and MutL proteins are conserved from bacteria to humans, and appear to be able to perform similar repair roles in higher organisms. For some of the lesions not well repaired by the MutS/MutL system, and for gene conversion where short-patch repair systems may be more desirable, other mismatch repair systems with novel capabilities are needed.
Currently, the most direct method for mutational analysis is DNA sequencing, however it is also the most labor intensive and expensive. It is usually not practical to sequence all potentially relevant regions of every experimental sample. Instead some type of preliminary screening method is commonly used to identify and target for sequencing only those samples that contain mutations. Single stranded conformational polymorphism (SSCP) is a widely used screening method based on mobility differences between single-stranded wild type and mutant sequences on native polyacrylamide gels. Other methods are based on mobility differences in wild type/mutant heteroduplexes (compared to control homoduplexes) on native gels (heteroduplex analysis) or denaturing gels (denaturing gradient gel electrophoresis). While sample preparation is relatively easy in these assays, very exacting conditions for electrophoresis are required to generate the often subtle mobility differences that form the basis for identifying the targets that contain mutations. Another critical parameter is the size of the target region being screened. In general, SSCP is used to screen target regions no longer than about 200-300 bases. The reliability of SSCP for detecting single-base mutations is somewhat uncertain but is probably in the 70-90% range for targets less than 200 bases. As the size of the target region increases, the detection rate declines, for example in one study from 87% for 183 bp targets to 57% for targets 307 bp in length (35). The ability to screen longer regions in a single step would enhance the utility of any mutation screening method.
Another type of screening technique currently in use is based on cleavage of unpaired bases in heteroduplexes formed between wild type probes hybridized to experimental targets containing point mutations. The cleavage products are also analyzed by gel electrophoresis, as subfragments generated by cleavage of the probe at a mismatch generally differ significantly in size from full length, uncleaved probe and are easily detected with a standard gel system. Mismatch cleavage has been effected either chemically (osmium tetroxide, hydroxylamine) or with a less toxic, enzymatic alternative, using RNase A. The RNase A cleavage assay has also been used, although much less frequently, to screen for mutations in endogenous mRNA targets or for detecting mutations in DNA targets amplified by PCR. A mutation detection rate of over 50% was reported for the original RNase screening method (36).
A newer method to detect mutations in DNA relies on DNA ligase which covalently joins two adjacent oligonucleotides which are hybridized on a complementary target nucleic acid. The mismatch must occur at the site of ligation. As with other methods that rely on oligonucleotides, salt concentration and temperature at hybridization are crucial. Another consideration is the amount of enzyme added relative to the DNA concentration.
The methods mentioned above cannot reliably detect a base change in a nucleic acid which is contaminated with more than 80% of a background nucleic acid, such as normal or wild type sequences. Contamination problems are significant in cancer detection wherein a malignant cell, in circulation for example, is present in extremely low amounts. The methods now in use lack adequate sensitivity to be practically applied in the clinical setting.
A method for the detection of gene mutations with mismatch repair enzymes has been described by Lu-Chang and Hsu. See WO 93/20233. The product of the MutY gene which recognizes mispaired A/G residues is employed in conjunction with another enzyme described in the reference as an xe2x80x9call type enzymexe2x80x9d which can nick at all base pair mismatches. The enzyme does not detect insertions and deletions. Also, the all type enzyme recognizes different mismatches with differing efficiencies and its activity can be adversely affected by flanking DNA sequences. This method therefore relies on a cocktail of mismatch repair enzymes and/or combinations of DNA glycosylases to detect the variety of mutations that can occur in a given DNA molecule.
The present invention provides materials and methods for the detection of mutations or mismatches in a targeted polynucleotide strand. Nucleic acid molecules encoding a mismatch endonuclease and methods of use thereof are disclosed. Detection is achieved using an endonuclease encoded by the nucleic acid molecules of the invention in combination with a gel assay system that facilitates the screening and identification of altered base pairing in a targeted nucleic acid strand. The availability of the nucleic acid having the sequence of SEQ ID NO:1 facilitates the preparation of large amounts of purified CEL I enzyme for use in such an assay.
In a preferred embodiment of the invention, an isolated nucleic acid molecule having the sequence of SEQ ID NO:1 encoding an endonuclease protein from celery about 43 kDa and 309 amino acids in length is provided. The endonuclease protein comprises a plurality of xcex1 helical domains and a flexible carboxy terminal region. The nucleic acid may be DNA or cDNA.
DNA molecules for isolating genomic clones of the invention are also provided. Such sequences facilitate the identification and cloning of a CEL I gene comprising introns and exons, the exons encoding the CEL 1 protein and specifically hybridizing with the nucleic acid of SEQ ID NO:1. Isolated RNA molecules transcribed from the nucleic acid of SEQ ID NO: 1 are also within the scope of the present invention.
In another aspect of the invention, a polynucleotide which comprises a) a sequence encoding a protein or polypeptide having SEQ ID NO: 2; b) a sequence encoding the complementary sequence of a); b) a sequence of nucleotides shown in FIG. 2; and c) a fragment of any of the sequences in a), or b) is disclosed.
In a preferred embodiment of the invention, an oligonucleotide between about 10 and about 200 nucleotides in length, which specifically hybridizes with SEQ ID NO:1 is provided.
In yet another aspect, an antibody immunologically specific for the isolated CEL I protein is provided. The antibody may be monoclonal or polyclonal.
Plasmids and vectors comprising SEQ ID NO: 1 are also within the scope of the present invention. In one embodiment, the vector may be a retroviral vector.
In a preferred embodiment of the invention, the plasmids or vectors described above may be introduced into host cells. Host cells suitable for this purpose include, without limitation, bacterial cells, plant cells, insect cells, procaryotic cells, fungal and mammalian cells.
Transgenic animals comprising SEQ ID NO: 1 are included in the present invention and have utility for assessing CEL I activities in a mammalian milieu.
Methods employing the nucleic acids of the invention are also provided. In one embodiment, a method for screening test compounds for CEL I modulating activity are provided. A host cell expressing a CEL I encoding nucleic acid is provided. The host cell is then contacted with a compound suspected of modulating CEL I activity and CEL I modulating activity is assessed by an alteration in the endonuclease activity of CEL I.
In a particularly preferred embodiment of the invention, a method for determining a mutation in a target sequence of single stranded polynucleotide with reference to a non-mutated sequence of a polynucleotide that is hybridizable with the polynucleotide including the target sequence is disclosed. The sequences are amplified, labeled with a detectable marker, hybridized to one another, exposed to a plant endonuclease encoded by a nucleic acid molecule having greater than  greater than 60% identity to a nucleic acid having the sequence of SEQ ID NO: 1, and analyzed for the presence of the mutation. In an alternative embodiment, the endonuclease is CEL I and is encoded by SEQ ID NO: 1. The availability of a nucleic acid having a sequence of SEQ ID NO: 1 facilitates the production of large quantities of the CEL I endonuclease for use in the method above. Exemplary endonucleases having greater than 60% sequence identity to CEL I are encoded by ZEN1 from Zinnia, BFN1 from Arabidopis and DSA6 from daylily.
Mismatch-specific nucleases corresponding to CEL I have been detected in more than 14 plant species. It is therefore anticipated that many additional plants contain nuclease genes that produce a protein with a high percentage of identity to SEQ ID NO:2. This use of these ortholog nuclease sequences to produce CEL I-like activity is contemplated with regard to the present invention. The encoded CEL I nuclease and its orthologs possess the following activities: i) detection of all mismatches between said hybridized sequences; ii) recognition of sequence differences in polynucleotide strands between about 100 bp and about 3 kb in length; and iii) recognition of said mutation in a target polynucleotide sequence without substantial adverse effect caused by flanking polynucleotide sequences.
DNA molecules and cDNA molecules may be assessed in the method described above. The method may be used to advantage in the screening assays for identifying alterations in DNA associated with genetic diseases and predisposition to cancer.
In yet another embodiment of the invention, an isozyme of CEL I having endonuclease activity is provided. The CEL I isozyme has a molecular weight of 39 kd and is isolated from celery.
In order to more clearly set forth the parameters of the present invention, the following definitions are used:
The term xe2x80x9cendonucleasexe2x80x9d refers to an enzyme that can cleave DNA internally.
The term xe2x80x9cbase pair mismatchxe2x80x9d indicates a base pair combination that generally does not form in nucleic acids according to Watson and Crick base pairing rules. For example, when dealing with the bases commonly found in DNA, namely adenine, guanine, cytosine and thymidine, base pair mismatches are those base combinations other than the A-T and G-C pairs normally found in DNA. As described herein, a mismatch may be indicated, for example as C/C meaning that a cytosine residue is found opposite another cytosine, as opposed to the proper pairing partner, guanine.
The phrase xe2x80x9cDNA insertion or deletionxe2x80x9d refers to the presence or absence of xe2x80x9cmatchedxe2x80x9d bases between two strands of DNA such that complementarity is not maintained over the region of inserted or deleted bases.
The term xe2x80x9ccomplementaryxe2x80x9d refers to two DNA strands that exhibit substantial normal base pairing characteristics. Complementary DNA may contain one or more mismatches, however.
The phrase xe2x80x9cflanking nucleic acid sequencesxe2x80x9d refers to those contiguous nucleic acid sequences that are 5xe2x80x2 and 3xe2x80x2 to the endonuclease cleavage site.
The term xe2x80x9cmultiplex analysisxe2x80x9d refers to the simultaneous assay of pooled DNA samples according to the above described methods.
C greater than T indicates the substitution of a thymidine residue for a cytosine residue giving rise to a mismatch. Inappropriate substitution of any base for another giving rise to a mismatch or a polymorphism may be indicated this way.
N, N, Nxe2x80x2, Nxe2x80x2-tetramethyl-6-carboxyrhodamine (TAMRA) is a fluorescent dye used to label DNA molecular weight standards which are in turn utilized as an internal standard for DNA analyzed by automated DNA sequencing.
Primers may be labeled fluorescently with 6-carboxyfluorescein (6-FAM). Alternatively primers may be labeled with 4, 7, 2xe2x80x2, 7xe2x80x2-Tetrachloro-6-carboxyfluorescein (TET). Other alternative DNA labeling methods are known in the art and are contemplated to be within the scope of the invention.
xe2x80x9cNucleic acidxe2x80x9d or a xe2x80x9cnucleic acid moleculexe2x80x9d as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5xe2x80x2 to 3xe2x80x2 direction. With reference to nucleic acids of the invention, the term xe2x80x9cisolated nucleic acidxe2x80x9d is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an xe2x80x9cisolated nucleic acidxe2x80x9d may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.
When applied to RNA, the term xe2x80x9cisolated nucleic acidxe2x80x9d refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
xe2x80x9cNatural allelic variantsxe2x80x9d, xe2x80x9cmutantsxe2x80x9d and xe2x80x9cderivativesxe2x80x9d of particular sequences of nucleic acids refer to nucleic acid sequences that are closely related to a particular sequence but which may possess, either naturally or by design, changes in sequence or structure. By closely related, it is meant that at least about 60%, but often, more than 85%, of the nucleotides of the sequence match over the defined length of the nucleic acid sequence referred to using a specific SEQ ID NO. Changes or differences in nucleotide sequence between closely related nucleic acid sequences may represent nucleotide changes in the sequence that arise during the course of normal replication or duplication in nature of the particular nucleic acid sequence. Other changes may be specifically designed and introduced into the sequence for specific purposes, such as to change an amino acid codon or sequence in a regulatory region of the nucleic acid. Such specific changes may be made in vitro using a variety of mutagenesis techniques or produced in a host organism placed under particular selection conditions that induce or select for the changes. Such sequence variants generated specifically may be referred to as xe2x80x9cmutantsxe2x80x9d or xe2x80x9cderivativesxe2x80x9d of the original sequence.
The terms xe2x80x9cpercent similarityxe2x80x9d, xe2x80x9cpercent identityxe2x80x9d and xe2x80x9cpercent homologyxe2x80x9d when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program and are further discussed below.
The present invention also includes active portions, fragments, derivatives and functional or non-functional mimetics of CEL I polypeptides or proteins of the invention. An xe2x80x9cactive portionxe2x80x9d of CEL I polypeptide means a peptide that is less than the full length CEL I polypeptide, but which retains measurable biological activity.
A xe2x80x9cfragmentxe2x80x9d or xe2x80x9cportionxe2x80x9d of the CEL I polypeptide means a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to thirteen contiguous amino acids and, most preferably, at least about twenty to thirty or more contiguous amino acids. A xe2x80x9cderivativexe2x80x9d of the CEL I polypeptide or a fragment thereof means a polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion or substitution of one or more amino acids, and may or may not alter the essential activity of the original CEL I polypeptide.
Different xe2x80x9cvariantsxe2x80x9d of the CEL I polypeptide exist in nature. These variants may be alleles characterized by differences in the nucleotide sequences of the gene coding for the protein, or may involve different RNA processing or post-translational modifications. The skilled person can produce variants having single or multiple amino acid substitutions, deletions, additions or replacements. These variants may include inter alia: (a) variants in which one or more amino acids residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the CEL I polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the CEL I polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the CEL I polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like. Other CEL I polypeptides of the invention include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or non-conserved positions. In another embodiment, amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques are known to the person having ordinary skill in the art.
To the extent such allelic variations, analogues, fragments, derivatives, mutants, and modifications, including alternative nucleic acid processing forms and alternative post-translational modification forms result in derivatives of the CEL I polypeptide that retain any of the biological properties of the CEL I polypeptide, they are included within the scope of this invention.
The term xe2x80x9corthologsxe2x80x9d as used herein refers to nucleases encoded by nucleic acid sequences whose polypeptide product has greater than 60% identity to the CEL I encoding sequence and whose gene products have similar three dimensional structure and biochemical activities of CEL I. The use of nucleases encoded by such orthologs in the methods of the invention is contemplated herein. Exemplary orthologs include, without limitation, ZEN1, BFN1 and DSA6.
The term xe2x80x9cfunctionalxe2x80x9d as used herein implies that the nucleic or amino acid sequence is functional for the recited assay or purpose.
The phrase xe2x80x9cconsisting essentially ofxe2x80x9d when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID No:. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.
A xe2x80x9crepliconxe2x80x9d is any genetic element, for example, a plasmid, cosmid, bacmid, phage or virus, that is capable of replication largely under its own control. A replicon may be either RNA or DNA and may be single or double stranded.
A xe2x80x9cvectorxe2x80x9d is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.
An xe2x80x9cexpression operonxe2x80x9d refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
The term xe2x80x9coligonucleotide,xe2x80x9d as used herein refers to primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.
The term xe2x80x9cprobexe2x80x9d as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The probes herein are selected to be xe2x80x9csubstantiallyxe2x80x9d complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to xe2x80x9cspecifically hybridizexe2x80x9d or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5xe2x80x2 or 3xe2x80x2 end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.
The term xe2x80x9cspecifically hybridizexe2x80x9d refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed xe2x80x9csubstantially complementaryxe2x80x9d). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
The term xe2x80x9cprimerxe2x80x9d as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3xe2x80x2 terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield an primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3xe2x80x2 hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5xe2x80x2 end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
The term xe2x80x9cisolated proteinxe2x80x9d or xe2x80x9cisolated and purified proteinxe2x80x9d is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in xe2x80x9csubstantially purexe2x80x9d form. xe2x80x9cIsolatedxe2x80x9d is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into, for example, immunogenic preparations or pharmaceutically acceptable preparations.
The term xe2x80x9csubstantially purexe2x80x9d refers to a preparation comprising at least 50-60% by weight of a given material (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-95% by weight of the given compound. Purity is measured by methods appropriate for the given compound (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
xe2x80x9cMature proteinxe2x80x9d or xe2x80x9cmature polypeptidexe2x80x9d shall mean a polypeptide possessing the sequence of the polypeptide after any processing events that normally occur to the polypeptide during the course of its genesis, such as protoelytic processing from a polyprotein precursor. In designating the sequence or boundaries of a mature protein, the first amino acid of the mature protein sequence is designated as amino acid residue 1.
The term xe2x80x9ctag,xe2x80x9d xe2x80x9ctag sequencexe2x80x9d or xe2x80x9cprotein tagxe2x80x9d refers to a chemical moiety, either a nucleotide, oligonucleotide, polynucleotide or an amino acid, peptide or protein or other chemical, that when added to another sequence, provides additional utility or confers useful properties, particularly in the detection or isolation, to that sequence. Thus, for example, a homopolymer nucleic acid sequence or a nucleic acid sequence complementary to a capture oligonucleotide may be added to a primer or probe sequence to facilitate the subsequent isolation of an extension product or hybridized product. In the case of protein tags, histidine residues (e.g., 4 to 8 consecutive histidine residues) may be added to either the amino- or carboxy-terminus of a protein to facilitate protein isolation by chelating metal chromatography. Alternatively, amino acid sequences, peptides, proteins or fusion partners representing epitopes or binding determinants reactive with specific antibody molecules or other molecules (e.g., flag epitope, c-myc epitope, transmembrane epitope of the influenza A virus hemaglutinin protein, protein A, cellulose binding domain, calmodulin binding protein, maltose binding protein, chitin binding domain, glutathione S-transferase, and the like) may be added to proteins to facilitate protein isolation by procedures such as affinity or immunoaffinity chromatography. Chemical tag moieties include such molecules as biotin, which may be added to either nucleic acids or proteins and facilitates isolation or detection by interaction with avidin reagents, and the like. Numerous other tag moieties are known to, and can be envisioned by, the trained artisan, and are contemplated to be within the scope of this definition.
The terms xe2x80x9ctransformxe2x80x9d, xe2x80x9ctransfectxe2x80x9d, xe2x80x9ctransducexe2x80x9d, shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion and the like.
The introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism. In bacterial, yeast, plant and mammalian cells, for example, the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid. Alternatively, the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell or organism. In other manners, the introduced nucleic acid may exist in the recipient cell or host organism only transiently.
A xe2x80x9cclonexe2x80x9d or xe2x80x9cclonal cell populationxe2x80x9d is a population of cells derived from a single cell or common ancestor by mitosis.
A xe2x80x9ccell linexe2x80x9d is a clone of a primary cell or cell population that is capable of stable growth in vitro for many generations.
An xe2x80x9cimmune responsexe2x80x9d signifies any reaction produced by an antigen, such as a protein antigen, in a host having a functioning immune system. Immune responses may be either humoral in nature, that is, involve production of immunoglobulins or antibodies, or cellular in nature, involving various types of B and T lymphocytes, dendritic cells, macrophages, antigen presenting cells and the like, or both. Immune responses may also involve the production or elaboration of various effector molecules such as cytokines, lymphokines and the like. Immune responses may be measured both in in vitro and in various cellular or animal systems.
An xe2x80x9cantibodyxe2x80x9d or xe2x80x9cantibody moleculexe2x80x9d is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. The term includes polyclonal, monoclonal, chimeric, and bispecific antibodies. As used herein, antibody or antibody molecule contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunloglobulin molecule such as those portions known in the art as Fab, Fabxe2x80x2, F(abxe2x80x2)2 and F(v).